A global mechanism of catabolite repression of the genus Bacillus comprises negative regulation exerted through the binding of the CcpA protein to the catabolite-responsive elements (cres) of the target genes. We searched for cue sequences in the Bacillus subtilis genome using a query sequence, WTGNAANCGNWNNCW (N and W stand for any base and A or T, respectively), picking out 126 putative and known cue sequences, To examine their cue function, we integrated spec promoter (Pspac)-cre-lacZ fusions into the amyE locus. Examination of catabolite repression of beta-galactosidase synthesis in the integrants led us to the following conclusions: (i) lower mismatching of cue sequences to the query sequence is required for their function; (ii) although cue sequences are partially palindromic, low mismatching in the same direction as that of transcription of the target genes is more critical for their function than that in the inverse direction; and (iii) yet, a more palindromic nature of cue sequences is desirable for a better function. Furthermore, the alignment of 22 cues that function in vivo implicated a consensus sequence, WWTGNAARCGNWWWCAWW (R stands for a or A). Interestingly, in the case where cue sequences are located in the protein-coding regions of the target genes, their conserved bases are preferentially the third bases of codons where base degeneracy is allowed.